[Study the capacity of recombinant HCV core+1 protein to induce immune responses in combination with different adjuvants]

Hepatitis C virus [HCV] genome contains a large open reading frame coding for a polyprotein that is cleaved into ten proteins. Recently, a new protein, named core+1, has been described to be expressed through a ribosomal frame shift within the capsid-encoding sequence. To address these possibilities, core+1 was produced in E.coli and the purified protein was evaluated for the immunological properties. The core+1 corresponding nucleotide sequence was created by PCR-based induction of a +1 frame shift mutation within the core gene template. The amplicon was cloned into the pET-24a vector and expressed in E.coli host. The expressed protein was purified under denaturing condition and after refolding steps was characterized by SDS-PAGE and Western Blotting. The immunization potential of core+1 with various adjuvant [Freunds [C/IFA], Montanide ISA 206 and IMS 1312, pluronic acid [F127] and imiquimod [IMIQ] was assessed in Balb/c mice. ELISA-based assays were used to analyze the humoral immune responses. The yield of E.coli-derived core+1 was 5 mg/ L of culture media and antigenicity of this protein was confirmed by western blotting. All the core+1/adjuvant formulations significantly developed the anti-core+1 IgG responses in the immunized mice but C/IFA and ISA206 elicited the highest antibody titers. ISA 206 and IMS 1312 formulation of core+1 induced strong Th1/Th2 responses. Our results indicated that core+1 formulation with various adjuvants may elicit the different immune response profiles [Th1/Th2]. Thus core+1 might be a potential component of future HCV vaccine too

[Designing and producing polytope DNA vaccine containing HBsAg gene for the induction of protective immunity against hepatitis C]

Background: considering the immunosuppressive effects and prevalent mutations in some HCV antigens, induction of CD8[+] T cell responses is focused on conserved and critical epitopes which as a multi-epitope vaccine can prevent the chronic nature of the disease

Materials and Methods: two immunodominant HLA-A2-restricted human epitopes [E2[614- 622] and NS3[1406-1415]] and two H-2[d]-restricted mouse epitopes [core[132-142] and E2[405-414]] were designed in a sequential tandem, predicted by immunoinformatic analyses. Following the synthesis, related nucleotide sequence was cloned into the pcDNA3.1 vector with and without the fusion of hepatitis B surface antigen [HBsAg]. Two constructed plasmids [pcDNA3.1.HPOL and pcDNA3.1.POL, respectively] were evaluated for the protein expression and secretion in Cos-7 cell line. After the vaccination of BALB/c mice [n=6 in each group] with different DNA and peptide immunization regimens, CD8[+] T cell activity as well as the type and protective potency of the induced responses were evaluated

Results: despite the induction of epitope-specific responses in pcDNA3.1.POL injected mice, the group immunized with pcDNA3.1.HPOL indicated a significant increase in the number and activity of CD8[+] T cells [P<0.05]. Peptide boosting of this group [formulated in two human-compatible adjuvant] still led to the more activation of CD8[+] cells, induction of Th1 response and the inhibition of tumor model growth [P<0.05]

Conclusion: fusion of HBsAg as a particle-forming sequence and the source of helper epitopes along the DNA-prime/peptide-boosting immunization regimen are proposed as two promising strategies to improve the CTL multi-epitope vaccines against HCV

Cloning, optimization of expression condition, purification and immunological characterization of hydrophilic section of HCV core Ag, expressed in E-coli by T7-ara BAD promoter

The capsid or core Ag of Hepatitis C virus is a multifunctional protein which has the principal pathogenesis and diagnostic role in HCV related infections and most of these properties are attributed to the hydrophilic section [amino acids 2-122] of this protein. For different research and diagnostic applications, high amounts of this protein in pure and original form are required. So, the aim of this study was to clone the gene, optimize the expression condition, purify it in the original form, and immunologically characterize hydrophilic section of HCV Core Ag, expressed by T7-araBAD promoter system in E.coli. The PCR amplified region corresponding to 2-122 section of this Ag from genotype lb was cloned in pIVEX 2.3, a T7 promoter derived vector. The proper construct after digestional analysis and sequencing confirmations was transformed into BL2 1 -Al E. coli, and protein expression under control of araBAD promoter by addition of 0.2% Arabinose was induced. After optimization of expression condition, purification of protein by NI-NTA agarose gel chromatography in native condition by immidazole yielded about 3.5mg/L of HCV core Ag. Immunological studies by western blotting through application of core specific mAbs and results of ELISA tests indicated that the protein is with desired immunological properties. AraBAD promoter can be perfectly utilized to produce the hydrophilic section of HCV core in high yields, and purification through NI-NTA in native condition may provide the antigen for different research and diagnostic applications

Evaluation of a native preparation of HCV core protein [2-122] for potential applications in immunization, diagnosis and mAb production

Infection with hepatitis C virus [HCV] is a worldwide problem. Among HCV proteins, core antigen [Ag], besides its importance for diagnostic application is a prime candidate for component of a vaccine. Herein, we report results of studies on production of the hydrophilic domain of core Ag [2-122] in native conformation by an arabinose induction system in E.coli and the primary characterization of this recombinant protein for applications in diagnosis, immunization and mAb production. Recombinant core [r-Core] was able to detect anti-core antibodies in HCV positive serum samples in a dilution rate of 1/3200. It was also capable to elicit a potent anti-HCV humoral immune response in BALB/c mice. Finally, we established two stable clones of hybridoma which shown to produce specific and sensitive mAbs against the core protein. HCV core was able to elicit a broad range of antibody specificities depending on the immunogen conformation. Therefore, it may be possible to get new mAbs with higher affinities towards native conformation of core Ag